Systemic inflammatory markers as diagnostic tools in the prevention of atherosclerotic diseases and as tools to aid in the selection of agents to be used for the prevention and treatment of atherosclerotic disease

ABSTRACT

The invention involves methods for characterizing an individual&#39;s risk profile of developing a future cardiovascular disorder by obtaining a level of the marker of systemic inflammation in the individual. The invention also involves methods for evaluating the likelihood that an individual will benefit from treatment with an agent for reducing the risk of future cardiovascular disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/387,028, filed on Aug. 31, 1999, now U.S. Pat. No. 7,030,152, issuedApr. 18, 2006, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/054,212 filed on Apr. 2, 1998, now U.S. Pat. No.6,040,147, issued Mar. 21, 2000, which in turn claims priority (under 35USC §119(e)) to U.S. provisional applications Ser. Nos. 60/043,039(filed Apr. 2, 1997), 60/041,950 (filed Apr. 2, 1997) and 60/070,894(filed Jan. 9, 1998), all entitled Systemic Inflammatory Markers asDiagnostic Tools in the Prevention of Atherosclerotic Diseases and asTools to Aid in the Selection of Agents to be Used for the Preventionand Treatment of Atherosclerotic Disease. The entire contents of allafore-mentioned applications are incorporated herein by reference.

GOVERNMENT SUPPORT

Some aspects of the present invention were made with support by grantsfrom the United States National Institutes of Health (NIH) under NIHgrants HL26490, HL34595, HL46696, CA34944, CA42182, and CA40360. TheU.S. Government retains certain rights in the invention.

FIELD OF THE INVENTION

This invention describes the new use of a diagnostic test to determinethe risk of atherosclerotic diseases such as myocardial infarction andstroke, particularly among individuals with no signs or symptoms ofcurrent disease and among nonsmokers. Further, this invention describesthe new use of a diagnostic test to assist physicians in determiningwhich individuals at risk will preferentially benefit from certaintreatments designed either to prevent first or recurrent myocardialinfarctions and strokes, or to treat acute and chronic cardiovasculardisorders.

BACKGROUND OF THE INVENTION

Despite significant advances in therapy, cardiovascular disease remainsthe single most common cause of morbidity and mortality in the developedworld. Thus, prevention of cardiovascular disorders such as myocardialinfarction and stroke is an area of major public health importance.Currently, several risk factors for future cardiovascular disorders havebeen described and are in wide clinical use in the detection ofindividuals at high risk. Such screening tests include evaluations oftotal and HDL cholesterol levels. However, a large number ofcardiovascular disorders occur in individuals with apparently low tomoderate risk profiles, and our ability to identify such patients islimited. Moreover, accumulating data suggests that the beneficialeffects of certain preventive and therapeutic treatments for patients atrisk for or known to have cardiovascular disorders differs in magnitudeamong different patient groups. At this time, however, data describingdiagnostic tests to determine whether certain therapies can be expectedto be more or less effective are lacking.

Certain cardiovascular disorders, such as myocardial infarction andischemic stroke, are associated with atherosclerosis. The mechanism ofatherosclerosis is not well understood. While inflammation ishypothesized to play a role in the initiation and progression ofatherosclerosis, clinical data have not established whether inflammationincreases, or anti-inflammatory treatments decrease, the risk ofcardiovascular disorders associated with atherosclerosis.

C-reactive protein is a marker for underlying systemic inflammation.Elevated levels of C-reactive protein have been described among patientswith acute ischemia or myocardial infarction, and predict episodes ofrecurrent ischemia among those hospitalized with unstable angina.Further, plasma concentration of C-reactive protein is associated withrisk of myocardial infarction among unhealthy patients, such as thosewith symptomatic angina pectoris. Plasma concentration of C-reactiveprotein also is associated with fatal, but not nonfatal, coronary heartdisease among smokers with multiple risk factors for atherosclerosis.However, since levels of C-reactive protein increase following acuteischemia and are directly related to cigarette consumption, it has beenuncertain whether statistical associations observed in these priorstudies of acutely ill or high-risk populations are causal, are due toshort-term inflammatory changes or are due to interrelations with otherrisk factors, in particular, smoking and hyperlipidemia.

SUMMARY OF INVENTION

This invention describes new diagnostic tests which determine andutilize the magnitude of systemic inflammation. These new tests broadlyinclude (1) the prediction of risk of future atherosclerotic disorderssuch as myocardial infarction and stroke and peripheral arterialdisease; and (2) the determination of the likelihood that certainindividuals will benefit to a greater or lesser extent from the use ofcertain treatments designed to prevent and/or treat atheroscleroticdisorders. These new tests are based in part upon the followingdiscoveries.

It has been discovered that elevated levels of markers of systemicinflammation are predictive of future cardiovascular disorders. Forexample, elevated levels of markers of systemic inflammation inapparently healthy, nonsmokers are predictive of an increased risk ofmyocardial infarction. As another example, contrary to suggestions inthe prior art, elevated levels of markers of systemic inflammation inotherwise healthy smokers are predictive of an increased risk of anonfatal myocardial infarction. As still another example, elevatedlevels of markers of systemic inflammation are predictive of anincreased likelihood of a future stroke.

It has been discovered also that the likelihood that certain individualswill benefit to a greater or a lesser extent from the use of certaintherapeutic agents for reducing the risk of a future cardiovasculardisorder can be determined from the base-line level of systemicinflammation in an individual.

It further has been discovered that the predictive value of markers ofsystemic inflammation are independent of other predictors and, forexample, are additive with risk factors derived from total cholesterollevels and total cholesterol/HDL ratios. Thus, the level of markers ofsystemic inflammation does not simply duplicate that which is measuredwhen levels of cholesterol are measured.

As mentioned above, these discoveries have led to new diagnostic tests.

Thus, according to one aspect of the invention, a method for treating asubject to reduce the risk of a cardiovascular disorder, is provided.The method involves selecting and administering to a subject who isknown to have an above-normal level of the marker of systemicinflammation an agent for reducing the risk of the cardiovasculardisorder. The agent can be an anti-inflammatory agent, an antithromboticagent, an anti-platelet agent, a fibrinolytic agent, a lipid reducingagent, a direct thrombin inhibitor, a glycoprotein IIb/IIIa receptorinhibitor, an agent that binds to cellular adhesion molecules andinhibits the ability of white blood cells to attach to such molecules, acalcium channel blocker, a beta-adrenergic receptor blocker, acyclooxygenase-2 inhibitor, an angiotensin system inhibitor, and/orcombinations thereof. The agent is administered in an amount effectiveto lower the risk of the subject developing a future cardiovasculardisorder. The preferred subjects are apparently healthy subjectsotherwise free of current need for treatment with any of the foregoingcategories of agents, for example, such as with respect toanti-inflammatory agents, free of symptoms of rheumatoid arthritis,chronic back pain, autoimmune diseases, and the like. In furtherimportant embodiments, the subject treated is a nonhyperlipidemicsubject. In another embodiment, the subjects are not at an elevated riskof an adverse cardiovascular event (e.g., subjects with no familyhistory of such events, subjects who are nonsmokers, subjects who arenonhyperlipidemic), other than having an elevated level of a marker ofsystemic inflammation. In other embodiments, the agent can be anon-aspirin, anti-inflammatory agent.

In certain embodiments, the agent is an anti-inflammatory agent selectedfrom the group consisting of Alclofenac; Alclometasone Dipropionate;Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; AmfenacSodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen;Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; BenzydamineHydrochloride; Bromelains; Broperamole; Budesonide; Carprofen;Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; ClobetasoneButyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate;Cortodoxone; Deflazacort; Desonide; Desoximetasone; DexamethasoneDipropionate; Diclofenac Potassium; Diclofenac Sodium; DiflorasoneDiacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone;Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium;Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone;Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin;Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin;Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate;Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide;Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;Triclonide; Triflumidate; Zidometacin; Glucocorticoids or ZomepiracSodium.

The invention also involves a method for treating subjects with a lipidreducing agent, to prevent cardiovascular disorders. Such an agent isadministered to a subject selected on the basis of having anabove-normal level of a marker of systemic inflammation. The agent isadministered in an amount effective to lower the risk of the subjectdeveloping a future cardiovascular disorder. In one embodiment, thesubject already has had a cardiovascular event, such as a heart attackor an angioplasty. In this embodiment, the lipid reducing agent canlimit further injury or help prevent restenosis, post-myocardialinfarction or post-angioplasty injury. In another important embodiment,the subjects are apparently healthy subjects otherwise free of currentneed for lipid reducing agent treatment. In further importantembodiments, the subject treated is a nonhyperlipidemic subject. In anyof the foregoing embodiments, the lipid reducing agent is gemfibrozil,cholystyramine, colestipol, nicotinic acid, probucol lovastatin,fluvastatin, simvastatin, atorvastatin, pravastatin, or cirivastatin. Inpreferred embodiments, the lipid reducing agent is pravastatin.

The invention also involves a method for treating subjects with an agentthat binds to a cellular adhesion molecule and that inhibits the abilityof white blood cells to attach to such molecules, to preventcardiovascular disorders. Such an agent is administered to a subjectselected on the basis of having an above-normal level of a marker ofsystemic inflammation. The agent is administered in an amount effectiveto lower the risk of the subject developing a future cardiovasculardisorder. In one embodiment, the subject already has had acardiovascular event, such as a heart attack or an angioplasty. In thisembodiment, the agent that binds to a cellular adhesion molecule andthat inhibits the ability of white blood cells to attach to suchmolecules, can limit further injury or help prevent restenosis,post-myocardial infarction or post-angioplasty injury. In anotherimportant embodiment, the subjects are apparently healthy subjectsotherwise free of current need for treatment with an agent that binds toa cellular adhesion molecule and that inhibits the ability of whiteblood cells to attach to such molecules. In further importantembodiments, the subject treated is a nonhyperlipidemic subject.

The invention also involves a method for treating subjects with acalcium channel blocker, to prevent cardiovascular disorders. Such anagent is administered to a subject selected on the basis of having anabove-normal level of a marker of systemic inflammation. The agent isadministered in an amount effective to lower the risk of the subjectdeveloping a future cardiovascular disorder. In one embodiment, thesubject already has had a cardiovascular event, such as a heart attackor an angioplasty. In another important embodiment, the subjects areapparently healthy subjects otherwise free of current need for calciumchannel blocker treatment. In further important embodiments, the subjecttreated is a nonhyperlipidemic subject. In any of the foregoingembodiments, the calcium channel blocker can be selected from the groupconsisting of dihydropyridines, phenyl alkyl amines, and/orbenzothiazepines. In preferred embodiments, calcium channel blockersuseful according to the invention, include, but are not limited to,aminone, amlodipine, bencyclane, diltiazem, felodipine, fendiline,flunarizine, isradipine, nicardipine, nifedipine, nimodipine,perhexilene, gallopamil, tiapamil and tiapamil analogues (such as1993RO-11-2933), verapamil, phenyloin, barbiturates, and the peptidesdynorphin, omega-conotoxin, and omega-agatoxin, and the like and/orpharmaceutically acceptable salts thereof.

The invention also involves a method for treating subjects with abeta-adrenergic receptor blocker, to prevent cardiovascular disorders.Such an agent is administered to a subject selected on the basis ofhaving an above-normal level of a marker of systemic inflammation. Theagent is administered in an amount effective to lower the risk of thesubject developing a future cardiovascular disorder. In one embodiment,the subject already has had a cardiovascular event, such as a heartattack or an angioplasty. In another important embodiment, the subjectsare apparently healthy subjects otherwise free of current need forbeta-adrenergic receptor blocker treatment. In further importantembodiments, the subject treated is a nonhyperlipidemic subject. In anyof the foregoing embodiments, the beta-adrenergic receptor blocker isselected from the group consisting of atenolol, acebutolol, alprenolol,befunolol, betaxolol, bunitrolol, carteolol, celiprolol, hedroxalol,indenolol, labetalol, levobunolol, mepindolol, methypranol, metindol,metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl, 1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxypropylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy) phthalide.

The invention also involves a method for treating subjects with acyclooxygenase-2 inhibitor, to prevent cardiovascular disorders. Such anagent is administered to a subject selected on the basis of having anabove-normal level of a marker of systemic inflammation. The agent isadministered in an amount effective to lower the risk of the subjectdeveloping a future cardiovascular disorder. In one embodiment, thesubject already has had a cardiovascular event, such as a heart attackor an angioplasty. In another important embodiment, the subjects areapparently healthy subjects otherwise free of current need forcyclooxygenase-2 inhibitor treatment. In further important embodiments,the subject treated is a nonhyperlipidemic subject. In any of theforegoing embodiments, the cyclooxygenase-2 inhibitor is selected fromthe group consisting of a phenyl heterocycle, a diaryl bicyclicheterocycle, an aryl substituted 5,5 fused aromatic nitrogen compound, aN-benzylindol-3-yl propanoic acid and/or its derivatives, a5-methanesulfonamido-1-indanone, a N-benzyl indol-3-yl butanoic acidand/or its derivatives, a diphenyl-1,2-3-thiadiazole, adiaryl-5-oxygenated-2-(5H)-furanone, a3,4-diaryl-2-hydroxy-2,5-dihydrofuran, a stilbene and/or itsderivatives, a diphenyl stilbene, an alkylated styrene, a bisarylcyclobutene and/or its derivatives, a substituted pyridine, apyridinyl-2-cyclopenten-1-one, and/or a substitutedsulfonylphenylheterocycle.

The invention also involves a method for treating subjects with anangiotensin system inhibitor, to prevent cardiovascular disorders. Suchan agent is administered to a subject selected on the basis of having anabove-normal level of a marker of systemic inflammation. The agent isadministered in an amount effective to lower the risk of the subjectdeveloping a future cardiovascular disorder. In one embodiment, thesubject already has had a cardiovascular event, such as a heart attackor an angioplasty. In another important embodiment, the subjects areapparently healthy subjects otherwise free of current need forangiotensin system inhibitor treatment. In further importantembodiments, the subject treated is a nonhyperlipidemic subject. In anyof the foregoing embodiments, the angiotensin system inhibitor inhibitoris selected from the group consisting of an angiotensin-convertingenzyme (ACE) inhibitor, an angiotensin II antagonist, an angiotensin IIreceptor antagonist, agents that activate the catabolism of angiotensinII, and/or agents that prevent the synthesis of angiotensin I.

According to another aspect of the invention, a method is provided forevaluating the likelihood that an individual will benefit from treatmentwith an agent for reducing the risk of a cardiovascular disorderassociated with atherosclerotic disease. The agent can be selected fromthe group consisting of anti-inflammatory agents, anti-thromboticagents, anti-platelet agents, fibrinolytic agents, lipid reducingagents, direct thrombin inhibitors, glycoprotein II b/IIIa receptorinhibitors, agents that bind to cellular adhesion molecules and inhibitthe ability of white blood cells to attach to such molecules (e.g.anti-cellular adhesion molecule antibodies), calcium channel blockers,beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors,angiotensin system inhibitors, and/or combinations of the foregoingagents thereof. A preferred agent is aspirin. To practice the method, alevel of a marker of systemic inflammation in an individual is obtained.This level then is compared to a predetermined value, wherein the levelof the marker of systemic inflammation in comparison to thepredetermined value is indicative of the likelihood that the individualwill benefit from treatment with the agent. The individual then can becharacterized in terms of the net benefit likely to be obtained bytreatment with the agent.

The predetermined value can be a single value, multiple values, a singlerange or multiple ranges. Thus, in one embodiment, the predeterminedvalue is a plurality of predetermined marker level ranges, and thecomparing step comprises determining in which of the predeterminedmarker level ranges the individual's level falls. In preferredembodiments, the individual is apparently healthy. In certainembodiments, the individual also is a nonsmoker. In preferredembodiments the marker of systemic inflammation is selected from thegroup consisting of C-reactive protein, a cytokine and a cellularadhesion molecule. In the a preferred embodiment, the marker of systemicinflammation is C-reactive protein. In another preferred embodiment, themarker of systemic inflammation is the cytokine IL-6. Particularlyuseful results have been obtained with both of these markers.

When the marker of systemic inflammation is C-reactive protein, then apreferred predetermined value is about 1¾ mg/l of blood. Anotherpreferred predetermined value is about 2 mg/l of blood. When ranges areemployed, it is preferred that one of the plurality of ranges be belowabout 1¾ mg/l of blood and that another of the ranges be above about 1¾mg/l of blood. When the marker of systemic inflammation is sICAM-1, acellular adhesion molecule, then a preferred predetermined value isabout 250 ng/ml of blood. The predetermined value will depend, ofcourse, on the particular marker selected and even upon thecharacteristics of the patient population in which the individual lies,described in greater detail below.

As mentioned above, the invention is particularly adapted to determiningwhich individuals will preferentially benefit from treatment with anagent for reducing the risk in the individuals of a cardiovasculardisorder such as a future stroke or a future myocardial infarction,including nonfatal myocardial infarctions. It also permits selection ofcandidate populations for clinical trials and for treatment withcandidate drugs, by identifying, for example, the individuals mostlikely to benefit from a new treatment or from a known treatment with ahigh risk profile of adverse side effects. Thus, the invention providesinformation for evaluating the likely net benefit of certain treatmentsfor candidate patients.

According to another aspect of the invention, a method is provided forcharacterizing an apparently healthy, nonsmoking individual's riskprofile of developing a future myocardial infarction. The methodinvolves obtaining a level of a marker of systemic inflammation in theindividual. The level of the marker then is compared to a predeterminedvalue, and the individual's risk profile of developing a futuremyocardial infarction then is characterized based upon the level of themarker in comparison to the predetermined value. As in the previousaspect of the invention, the predetermined value may be a single value,a plurality of values, a single range or a plurality of ranges. In oneembodiment, the predetermined value is a plurality of predeterminedmarker level ranges and the comparing step involves determining in whichof the predetermined marker level ranges the individual's level falls.The preferred markers, predetermined values and the like are asdescribed above.

According to still another aspect of the invention, a method is providedfor characterizing an individual's risk profile of developing a futurecardiovascular disorder associated with atherosclerotic disease, otherthan fatal myocardial infarction. A level of a marker of systemicinflammation in the individual is obtained. The level of the marker iscompared to a predetermined value. The individual's risk profile ofdeveloping the future cardiovascular disorder associated withatherosclerotic disease, other than a fatal cardiovascular event, thenis characterized based upon the level of the marker in comparison to thepredetermined value. The predetermined value can be as described above.The individual characterized may be any individual, but preferably is anapparently healthy individual. The apparently healthy individual can bea smoker or a nonsmoker. The preferred markers and predetermined valuesare as described above. In one important embodiment, the cardiovasculardisorder is stroke. In another important embodiment, the cardiovasculardisorder is nonfatal myocardial infarction. In another importantembodiment, the cardiovascular disorder is peripheral artery disease.

According to yet another aspect of the invention, a method is providedin which one uses an inflammatory marker together with a cholesterolfraction for characterizing an individual's risk profile of developing afuture cardiovascular disorder associated with atherosclerotic disease.A level of a marker of systemic inflammation in the individual isobtained. The level of the marker is compared to a predetermined valueto establish a first risk value. A level of a cholesterol in theindividual also is obtained. The level of the cholesterol in theindividual is compared to a second predetermined value to establish asecond risk value. The individual's risk profile of developing thecardiovascular disorder then is characterized based upon the combinationof the first risk value and the second risk value, wherein thecombination of the first risk value and second risk value establishes athird risk value different from the first and second risk values. Inparticularly important embodiments, the third risk value is greater thaneither of the first and second risk values. The preferred individualsfor testing, markers and predetermined values are as described above.The cardiovascular disorder can be any cardiovascular disorderassociated with atherosclerotic disease, although in certain importantembodiments the cardiovascular disorder is nonfatal myocardialinfarction or ischemic stroke.

The invention also contemplates kits comprising a package including anassay for a marker of systemic inflammation and instructions, andoptionally related materials such as number or color charts, forcorrelating the level of the marker as determined by the assay with arisk of developing a future cardiovascular disorder or with otherpatient criteria as described above. In important embodiments, the kitsalso include an assay for a cholesterol.

These and other aspects of the invention will be described in moredetail below in connection with the detailed description of theinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph demonstrating the relative risk of first myocardialinfarction in the study population according to baseline level ofC-reactive protein. Data are shown for all study subjects and/ornon-smokers.

FIG. 2 is a graph demonstrating the relative risks of future myocardialinfarction associated with high, middle and low tertiles of totalcholesterol and C-reactive protein.

FIG. 3 is a graph demonstrating the relative risks of future myocardialinfarction associated with high, middle and low tertiles of totalcholesterol:HDL cholesterol ratio and C-reactive protein.

FIG. 4 is a graph demonstrating relative risks (and 95 percentconfidence interval) of first myocardial infarction associated with eachincreasing quartile of baseline C-reactive protein, according to year ofstudy follow-up.

FIG. 5 is a graph demonstrating relative risks of first myocardialinfarction associated with baseline levels of C-reactive protein,stratified by randomized assignment to aspirin or placebo therapy.Analyses are limited to events occurring prior to unblinding of theaspirin component of the Physicians' Health Study. The reduction in riskof myocardial infarction associated with aspirin use was 13.9 percent inthe first (lowest) quartile of C-reactive protein, 33.4 percent in thesecond quartile, 46.3 percent in the third quartile, and 55.7 percent inthe fourth (highest) quartile.

FIG. 6 is a graph demonstrating the distribution of levels of C-reactiveprotein in the population studied in Example. 1.

FIG. 7 is a graph demonstrating the normal bell curve distribution whichoccurs when the C-reactive protein levels of FIG. 5 are log normalized.

DETAILED DESCRIPTION

The primary basis for this invention is evidence from the Physicians'Health Study, a large scale, randomized, double-blind, placebocontrolled trial of aspirin and beta-carotene in the primary preventionof cardiovascular disease conducted among 22,000 apparently healthy men.In that trial, baseline level of C reactive protein, a marker forunderlying systemic inflammation, was found to determine the future riskof myocardial infarction and stroke, independent of a large series oflipid and non-lipid risk factors. Specifically, individuals with thehighest baseline levels of C-reactive protein were found to have 3 foldincreases in risk of developing future myocardial infarction and 2 foldincreases in risk of developing future stroke. (FIG. 1).

Moreover, in data from the Physicians' Health Study, the risk of futuremyocardial infarction and stroke associated with this marker ofinflammation appear to be additive to that which could otherwise bedetermined by usual assessment of total cholesterol and HDL cholesterol.In this trial, the predictive value of C-reactive protein was presentfor non-fatal as well as fatal events, was stable over long periods oftime, and was present for non-smokers as well as smokers. Further, datafrom this trial indicate that the magnitude of benefit that apparentlyhealthy individuals can expect from prophylactic aspirin is dependent inlarge part upon baseline level of C-reactive protein. In addition, thesedata indicate that the benefit of other therapeutic agents used in theprevention and treatment of atherosclerotic disorders may differdepending on the underlying level of C-reactive protein. These data alsoraised the possibility that other inflammatory markers may have animportant role in determining the risk of myocardial infarction andstroke. This was tested. Data deriving from this study with regard toanother marker of inflammation, plasma level of the soluble cellularadhesion molecule sICAM-1, indicate the ability of other inflammatorymarkers to predict atherosclerotic risk.

The current invention in one aspect describes the use of inflammatorymarkers to predict risk of cardiovascular disorders associated withatherosclerosis such as myocardial infarction and stroke amongindividuals without current evidence of disease. Thus, these datagreatly extend prior observations regarding the use of inflammatorymarkers such as C reactive protein to predict risk among alreadyidentified high-risk populations (such as smokers) or among symptomaticischemia patients such as those with stable and unstable anginapectoris. Indeed, since levels of C reactive protein and other acutephase reactants increase following acute ischemia and are directlyrelated to cigarette consumption, it has been uncertain whetherstatistical associations observed in prior studies of acutely ill orhigh-risk populations are casual or due to short-term inflammatorychanges, or to interrelations with other risk factors, in particularsmoking and hyperlipidemia.

In marked contrast, data from the Physicians' Health Study indicate forthe first time the utility of inflammatory markers to predict risk amongcurrently healthy and otherwise low-risk individuals, to predictnon-fatal as well as fatal events, to predict risk among non-smokers,and to predict risk above and beyond that associated with screening fortotal and HDL cholesterol. Data from the Physicians' Health Study alsoindicate for the first time that the efficacy of interventions designedto reduce risk of atherosclerotic events such as myocardial infarctionand stroke differs in magnitude based upon a measure of the extent ofunderlying systemic inflammation.

The invention will be better understood with reference to the followingbrief explanation of terms.

“Cardiovascular disorders associated with atherosclerotic disease”includes myocardial infarction, stroke, angina pectoris and peripheralarteriovascular disease. Cardiovascular disorders associated withatherosclerotic disease do not include venous thrombosis.

“Apparently healthy”, as used herein, means individuals who have notpreviously had an acute adverse cardiovascular event such as amyocardial infarction (i.e., individuals who are not at an elevated riskof a second adverse cardiovascular event due to a primary adversecardiovascular event). Apparently healthy individuals also do nototherwise exhibit symptoms of disease. In other words, such individuals,if examined by a medical professional, would be characterized as healthyand free of symptoms of disease.

According to one important aspect of the invention, a method fortreating a subject to reduce the risk of a cardiovascular disorder, isprovided. The method involves selecting and administering to a subjectwho is known to have an above-normal level of the marker of systemicinflammation an agent for reducing the risk of the cardiovasculardisorder. The agent can be an anti-inflammatory agent, an antithromboticagent, an anti-platelet agent, a fibrinolytic agent, a lipid reducingagent, a direct thrombin inhibitor, a glycoprotein IIb/IIIa receptorinhibitor, an agent that binds to cellular adhesion molecules andinhibits the ability of white blood cells to attach to such molecules, acalcium channel blocker, a beta-adrenergic receptor blocker, acyclooxygenase-2 inhibitor, an angiotensin system inhibitor, and/orcombinations thereof. The agent is administered in an amount effectiveto lower the risk of the subject developing a future cardiovasculardisorder. The preferred subjects are apparently healthy subjectsotherwise free of current need for treatment with any of the foregoingcategories of agents, such as, for example, with respect toanti-inflammatory agents, free of symptoms of rheumatoid arthritis,chronic back pain, autoimmune diseases, and the like. In furtherimportant embodiments, the subject treated is a nonhyperlipidemicsubject. In another embodiment, the subjects are not at an elevated riskof an adverse cardiovascular event (e.g., subjects with no familyhistory of such events, subjects who are nonsmokers, subjects who arenonhyperlipidemic), other than having an elevated level of a marker ofsystemic inflammation.

In some embodiments, the subject is otherwise free of symptoms callingfor treatment with any of the foregoing categories of agents. In certainembodiments, the subject treated is apparently healthy. In furtherimportant embodiments, the subject treated is a nonhyperlipidemicsubject. A “nonhyperlipidemic” is a subject that is anonhypercholesterolemic and/or a nonhypertriglyceridemic subject. A“nonhypercholesterolemic” subject is one that does not fit the currentcriteria established for a hypercholesterolemic subject. Anonhypertriglyceridemic subject is one that does not fit the currentcriteria established for a hypertriglyceridemic subject (See, e.g.,Harrison's Principles of Experimental Medicine, 13th Edition,McGraw-Hill, Inc., N.Y.—hereinafter “Harrison's”). Hypercholesterolemicsubjects and hypertriglyceridemic subjects are associated with increasedincidence of premature coronary heart disease. A hypercholesterolemicsubject has an LDL level of >160 mg/dL, or >130 mg/dL and at least tworisk factors selected from the group consisting of male gender, familyhistory of premature coronary heart disease, cigarette smoking (morethan 10 per day), hypertension, low HDL (<35 mg/dL), diabetes mellitus,hyperinsulinemia, abdominal obesity, high lipoprotein (a), and personalhistory of cerebrovascular disease or occlusive peripheral vasculardisease. A hypertriglyceridemic subject has a triglyceride (TG) levelof >250 mg/dL. Thus, a nonhyperlipidemic subject is defined as one whosecholesterol and triglyceride levels are below the limits set asdescribed above for both the hypercholesterolemic andhypertriglyceridemic subjects.

“Nonsmoking”, as used herein, means an individual who, at the time ofthe evaluation, is not a smoker. This includes individuals who havenever smoked as well as individuals who in the past have smoked butpresently no longer smoke.

Agents for reducing the risk of a cardiovascular disorder include thoseselected from the group consisting of anti-inflammatory agents,anti-thrombotic agents, anti-platelet agents, fibrinolytic agents, lipidreducing agents, direct thrombin inhibitors, glycoprotein II b/IIIareceptor inhibitors, agents that bind to cellular adhesion molecules andinhibit the ability of white blood cells to attach to such molecules(e.g. anti-cellular adhesion molecule antibodies), calcium channelblockers, beta-adrenergic receptor blockers, cyclooxygenase-2inhibitors, angiotensin system inhibitors, and/or any combinationsthereof.

One preferred agent is aspirin.

“Anti-inflammatory” agents include Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; CormethasoneAcetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab;Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac;Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate;Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone;Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam;Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; TolmetinSodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids;Zomepirac Sodium.

“Anti-thrombotic” and/or “fibrinolytic” agents include Plasminogen (toplasmin via interactions of prekallikrein, kininogens, Factors XII,XIIIa, plasminogen proactivator, and tissue plasminogen activator[TPA])Streptokinase; Urokinase: Anisoylated Plasminogen-StreptokinaseActivator Complex; Pro-Urokinase; (Pro-UK); rTPA (alteplase or activase;r denotes recombinant); rPro-UK; Abbokinase; Eminase; SreptaseAnagrelide Hydrochloride; Bivalirudin; Dalteparin Sodium; DanaparoidSodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium;Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; retaplase; Trifenagrel;Warfarin; Dextrans.

“Anti-platelet” agents include Clopridogrel; Sulfinpyrazone; Aspirin;Dipyridamole; Clofibrate; Pyridinol Carbamate; PGE; Glucagon;Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin; Ticlopidine;Anagrelide.

“Lipid reducing” agents include gemfibrozil, cholystyramine, colestipol,nicotinic acid, probucol lovastatin, fluvastatin, simvastatin,atorvastatin, pravastatin, cirivastatin.

“Direct thrombin inhibitors” include hirudin, hirugen, hirulog,agatroban, PPACK, thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” are both antibodies andnon-antibodies, and include but are not limited to ReoPro (abcixamab),lamifiban, tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v. 52, (suppl. 1), p. 13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogenous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels. (Remington, TheScience and Practice of Pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, amrinone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexilene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenyloin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxypropylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

Cyclooxygenase-2 (COX-2) is a recently identified new form of acyclooxygenase. “Cyclooxygenase” is an enzyme complex present in mosttissues that produces various prostaglandins and thromboxanes fromarachidonic acid. Non-steroidal, antiinflammatory drugs exert most oftheir antiinflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of the cyclooxygenase (also known as prostaglandinG/Hsynthase and/orprostaglandin-endoperoxide synthase). Initially, onlyone form of cyclooxygenase was known, the “constitutive enzyme” orcyclooxygenase-1 (COX-1). It and was originally identified in bovineseminal vesicles.

Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources (See, e.g., U.S. Pat.No. 5,543,297, issued Aug. 6, 1996 to Cromlish, et al., and assigned toMerck Frosst Canada, Inc., Kirkland, Calif., entitled: “Humancyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity”). This enzyme is distinct from the COX-1. COX-2, is rapidlyand readily inducible by a number of agents including mitogens,endotoxin, hormones, cytokines and growth factors. As prostaglandinshave both physiological and pathological roles, it is believed that theconstitutive enzyme, COX-1, is responsible, in large part, forendogenous basal release of prostaglandins and hence is important intheir physiological functions such as the maintenance ofgastrointestinal integrity and renal blood flow. By contrast, it isbelieved that the inducible form, COX-2, is mainly responsible for thepathological effects of prostaglandins where rapid induction of theenzyme would occur in response to such agents as inflammatory agents,hormones, growth factors, and cytokines. Therefore, it is believed thata selective inhibitor of COX-2 has similar antiinflammatory, antipyreticand analgesic properties to a conventional non-steroidalantiinflammatory drug, and in addition inhibits hormone-induced uterinecontractions and also has potential anti-cancer effects, but withreduced side effects. In particular, such COX-2 inhibitors are believedto have a reduced potential for gastrointestinal toxicity, a reducedpotential for renal side effects, a reduced effect on bleeding times andpossibly a decreased potential to induce asthma attacks inaspirin-sensitive asthmatic subjects, and are therefore useful accordingto the present invention.

A number of selective “COX-2 inhibitors” are known in the art. Theseinclude, but are not limited to, COX-2 inhibitors described in U.S. Pat.No. 5,474,995 “Phenyl heterocycles as cox-2 inhibitors”; U.S. Pat. No.5,521,213 “Diaryl bicyclic heterocycles as inhibitors ofcyclooxygenase-2”; U.S. Pat. No. 5,536,752 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,550,142 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,552,422 “Aryl substituted 5,5 fusedaromatic nitrogen compounds as anti-inflammatory agents”; U.S. Pat. No.5,604,253 “N-benzylindol-3-yl propanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-diaryl-2-hydroxy-2,5-dihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif.). Additional COX-2 inhibitors are alsodescribed in U.S. Pat. No. 5,643,933, assigned to G.D. Searle & Co.(Skokie, Ill.), entitled: “Substituted sulfonylphenylheterocycles ascyclooxygenase-2 and 5-lipoxygenase inhibitors.”

A number of the above-identified COX-2 inhibitors are prodrugs ofselective COX-2 inhibitors, and exert their action by conversion in vivoto the active and selective COX-2 inhibitors. The active and selectiveCOX-2 inhibitors formed from the above-identified COX-2 inhibitorprodrugs are described in detail in WO 95/00501, published Jan. 5, 1995,WO 95/18799, published Jul. 13, 1995 and U.S. Pat. No. 5,474,995, issuedDec. 12, 1995. Given the teachings of U.S. Pat. No. 5,543,297, entitled:“Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity,” a person of ordinary skill in the art would be able todetermine whether an agent is a selective COX-2 inhibitor or a precursorof a COX-2 inhibitor, and therefore part of the present invention.

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin I and angiotensin II are synthesized by the enzymaticrenin-angiotensin pathway. The synthetic process is initiated when theenzyme renin acts on angiotensinogen, a pseudoglobulin in blood plasma,to produce the decapeptide angiotensin I. Angiotensin I is converted byangiotensin converting enzyme (ACE) to angiotensin II (angiotensin-[1-8]octapeptide). The latter is an active pressor substance which has beenimplicated as a causative agent in several forms of hypertension invarious mammalian species, e.g., humans.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs,etc. Among the most potent and useful renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists. In a preferred embodiment of the invention, therenin-angiotensin system inhibitors are renin inhibitors, ACEinhibitors, and angiotensin II antagonists.

“Angiotensin II antagonists” are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin II antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹⁾(Val⁵)(Ala⁸)] angiotensin-(1-8) octapeptide and related analogs);N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634); imidazoleacetate derivatives including 2-N-butyl-4-chloro-1-(2-chlorobenzile)imidazole-5-acetic acid (see Long et al., J. Pharmacol. Exp. Ther.247(1), 1-7 (1988));4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid andanalog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazolebeta-glucuronide analogs (U.S. Pat. No. 5,085,992); substitutedpyrroles, pyrazoles, and tryazoles (U.S. Pat. No. 5,081,127); phenol andheterocyclic derivatives such as 1,3-imidazoles (U.S. Pat. No.5,073,566); imidazo-fused 7-member ring heterocycles (U.S. Pat. No.5,064,825); peptides (e.g., U.S. Pat. No. 4,772,684); antibodies toangiotensin II (e.g., U.S. Pat. No. 4,302,386); and aralkyl imidazolecompounds such as biphenyl-methyl substituted imidazoles (e.g., EPNumber 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(−1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl (35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566 (E-alpha-2-[2-butyl-1-(carboxyphenyl) methyl] 1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPontMerck Pharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRocheAG); A₂ agonists (Marion Merrill Dow) and certain non-peptideheterocycles (G.D. Searle and Company).

“Angiotensin converting enzyme (ACE), is an enzyme which catalyzes theconversion of angiotensin I to angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di and tripeptides and antibodies to ACE which intervene in the renin-angiotensinsystem by inhibiting the activity of ACE thereby reducing or eliminatingthe formation of pressor substance angiotensin II. ACE inhibitors havebeen used medically to treat hypertension, congestive heart failure,myocardial infarction and renal disease. Classes of compounds known tobe useful as ACE inhibitors include acylmercapto and mercaptoalkanoylprolines such as captopril (U.S. Pat. No. 4,105,776) and zofenopril(U.S. Pat. No. 4,316,906), carboxyalkyl dipeptides such as enalapril(U.S. Pat. No. 4,374,829), lisinopril (U.S. Pat. No. 4,374,829),quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S. Pat. No. 4,587,258),and perindopril (U.S. Pat. No. 4,508,729), carboxyalkyl dipeptide mimicssuch as cilazapril (U.S. Pat. No. 4,512,924) and benazapril (U.S. Pat.No. 4,410,520), phosphinylalkanoyl prolines such as fosinopril (U.S.Pat. No. 4,337,201) and trandolopril.

“Renin inhibitors” are compounds which interfere with the activity ofrenin. Renin inhibitors include amino acids and derivatives thereof,peptides and derivatives thereof, and antibodies to renin. Examples ofrenin inhibitors that are the subject of United States patents are asfollows: urea derivatives of peptides (U.S. Pat. No. 5,116,835); aminoacids connected by nonpeptide bonds (U.S. Pat. No. 5,114,937); di andtri peptide derivatives (U.S. Pat. No. 5,106,835); amino acids andderivatives thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diolsulfonamides and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides(U.S. Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No. 5,075,451);fluorine and chlorine statine or statone containing peptides (U.S. Pat.No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079); N-morpholino derivatives (U.S. Pat. No. 5,055,466);pepstatin derivatives (U.S. Pat. No. 4,980,283); N-heterocyclic alcohols(U.S. Pat. No. 4,885,292); monoclonal antibodies to renin (U.S. Pat. No.4,780,401); and a variety of other peptides and analogs thereof (U.S.Pat. Nos. 5,071,837, 5,064,965, 5,063,207, 5,036,054, 5,036,053,5,034,512, and 4,894,437).

Agents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology. Such antibodies alreadyare known in the art and include anti-ICAM 1 antibodies as well as othersuch antibodies. Significantly, as is well-known in the art, only asmall portion of an antibody molecule, the paratrope, is involved in thebinding of the antibody to its epitope (see, in general, Clark, W. R.(1986) The Experimental Foundations of Modern Immunology, Wiley & Sons,Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, forexample, are effectors of the complement cascade but are not involved inantigen binding. An antibody from which the pFc′ region has beenenzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. Similarly, an antibody from whichthe Fc region has been enzymatically cleaved, or which has been producedwithout the Fc region, designated an Fab fragment, retains one of theantigen binding sites of an intact antibody molecule. Proceedingfurther, Fab fragments consist of a covalently bound antibody lightchain and a portion of the antibody heavy chain denoted Fd. The Fdfragments are the major determinant of antibody specificity (a single FdFragment may be associated with up to ten different light chains withoutaltering antibody specificity) and Fd fragments retain epitope-bindingability in isolation.

Within the antigen-binding portion of an antibody, as is well-know inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(Frs), which maintain the tertiary structure of the paratope (see, ingeneral, Clar, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or Fr and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornonhuman sequences. The present invention also includes so-called singlechain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to cellular adhesion molecules. These polypeptides maybe derived also from sources other than antibody technology. Forexample, such polypeptide binding agents can be provided by degeneratepeptide libraries which can be readily prepared in solution, inimmobilized form or as phage display libraries. Combinatorial librariesalso can be synthesized of peptides containing one or more amino acids.Libraries further can be synthesized of peptoids and non-peptidesynthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe cellular adhesion molecule. This process can be repeated throughseveral cycles of reselection of phage that bind to the cellularadhesion molecule. Repeated rounds lead to enrichment of phage bearingparticular sequences. DNA sequences analysis can be conducted toidentify the sequences of the expressed polypeptides. The minimal linearportion of the sequence that binds to the cellular adhesion molecule canbe determined. One can repeat the procedure using a biased librarycontaining inserts containing part of all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Yeast two-hybrid screening methods also may be used to identifypolypeptides that bind to the cellular adhesion molecules. Thus,cellular adhesion molecules, or a fragment thereof, can be used toscreen peptide libraries, including phage display libraries, to identifyand select peptide binding partners of the cellular adhesion molecules.

In practicing the methods of the present invention, it is required toobtain a level of a marker of systemic inflammation in an individual.Markers of systemic inflammation are well-known to those of ordinaryskill in the art. It is preferred that the markers of systemicinflammation be selected from the group consisting of C-reactiveprotein, cytokines, and cellular adhesion molecules. Cytokines arewell-known to those of ordinary skill in the art and include humaninterleukins 1-17. A preferred cytokine useful as marker of systemicinflammation is IL-6. Cellular adhesion molecules are well-known tothose of ordinary skill in the art and include integrins, ICAM-1,ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, and PECAM. The preferred adhesionmolecule is soluble intercellular adhesion molecule (sICAM-1).

The level of the marker of systemic inflammation for the individual canbe obtained by any art recognized method. Typically, the level isdetermined by measuring the level of the marker in a body fluid, forexample, blood, lymph, saliva, urine and the like. The level can bedetermined by ELISA, or immunoassays or other conventional techniquesfor determining the presence of the marker. Conventional methods includesending samples of a patient's body fluid to a commercial laboratory formeasurement.

The invention also involves comparing the level of marker for theindividual with a predetermined value. The predetermined value can takea variety of forms. It can be single cut-off value, such as a median ormean. It can be established based upon comparative groups, such as wherethe risk in one defined group is double the risk in another definedgroup. It can be a range, for example, where the tested population isdivided equally (or unequally) into groups, such as a low-risk group, amedium-risk group and a high-risk group, or into quadrants, the lowestquadrant being individuals with the lowest risk and the highest quadrantbeing individuals with the highest risk.

The predetermined value can depend upon the particular populationselected. For example, an apparently healthy, nonsmoker population (nodetectable disease and no prior history of a cardiovascular disorder)will have a different ‘normal’ range of markers of systemic inflammationthan will a smoking population or a population the members of which havehad a prior cardiovascular disorder. Accordingly, the predeterminedvalues selected may take into account the category in which anindividual falls. Appropriate ranges and categories can be selected withno more than routine experimentation by those of ordinary skill in theart.

The preferred body fluid is blood and the preferred marker is C-reactiveprotein. For C-reactive protein, one important cut-off for a populationof apparently healthy, nonsmokers is 1.75 mg/liter (median). Anotherimportant cut-off for C-reactive protein is 2.0 mg/liter (highestquartile of risk). In characterizing risk, numerous predetermined valuescan be established. In the preferred embodiment employing C-reactiveprotein, the cut-off values described above, and in greater detail inthe example below, are surprisingly lower than those shown in the priorart where C-reactive protein levels are studied in unhealthy individualsor smokers.

There presently are commercial sources which produce reagents for assaysfor C-reactive protein. These include, but are not limited to, AbbottPharmaceuticals (Abbott Park, Ill.), CalBiochem (San Diego, Calif.) andBehringwerke (Marburg, Germany). Commercial sources for inflammatorycytokine and cellular adhesion molecule measurements, include, but arenot limited to, R&D Systems (Minneapolis, Minn.), Genzyme (Cambridge,Mass.) and Immunotech (Westbrook, Me.).

In preferred embodiments the invention provides novel kits or assayswhich are specific for, and have appropriate sensitivity with respectto, predetermined values selected on the basis of the present invention.The preferred kits, therefore, would differ from those presentlycommercially available, by including, for example, different cut-offs,different sensitivities at particular cut-offs as well as instructionsor other printed material for characterizing risk based upon the outcomeof the assay.

As discussed above the invention provides methods for evaluating thelikelihood that an individual will benefit from treatment with an agentfor reducing risk of a future cardiovascular disorder. This method hasimportant implications for patient treatment and also for clinicaldevelopment of new therapeutics. Physicians select therapeutic regimensfor patient treatment based upon the expected net benefit to thepatient. The net benefit is derived from the risk to benefit ratio. Thepresent invention permits selection of individuals who are more likelyto benefit by intervention, thereby aiding the physician in selecting atherapeutic regimen. This might include using drugs with a higher riskprofile where the likelihood of expected benefit has increased.Likewise, clinical investigators desire to select for clinical trials apopulation with a high likelihood of obtaining a net benefit. Thepresent invention can help clinical investigators select suchindividuals. It is expected that clinical investigators now will use thepresent invention for determining entry criteria for clinical trials.

In another surprising aspect of the invention, it has been discoveredthat markers of systemic inflammation have predictive value independentof other known predictors of future adverse cardiovascular disorders.Thus, the present invention does not involve simply duplicating ameasurement that previously could be made using other predictors.Instead, the markers of systemic inflammation are additive to prior artpredictors. This is illustrated in FIGS. 2 and 3, wherein the data ofthe present invention is analyzed to characterize the risk profiles ofindividuals, taking into account, both total cholesterol levels andlevels of C-reactive protein. FIG. 2 shows the relative risk of futuremyocardial infarction associated with high, middle and low tertiles oftotal cholesterol and C reactive protein. FIG. 3 shows similarly therelative risk of future myocardial infarction associated with high,middle and low tertiles of total cholesterol:HDL ratio and C reactiveprotein. As is abundantly clear, the risk is additive.

The invention also involves a method for treating subjects, withanti-inflammatory therapies, to prevent cardiovascular disorders. Anagent selected from the group consisting of an anti-inflammatory agent,an antithrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, or an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, and/or any combinations thereof, is administered to asubject who has an above-normal level of a marker of systemicinflammation. The agent is administered in an amount effective to lowerthe risk of the subject developing a future cardiovascular disorder. Insome embodiments the agent is a non-aspirin anti-inflammatory agent.Agents are described elsewhere herein.

An effective amount is a dosage of the anti-inflammatory agentsufficient to provide a medically desirable result. The effective amountwill vary with the particular condition being treated, the age andphysical condition of the subject being treated, the severity of thecondition, the duration of the treatment, the nature of the concurrenttherapy (if any), the specific route of administration and the likefactors within the knowledge and expertise of the health practioner. Forexample, an effective amount can depend upon the degree to which anindividual has abnormally elevated levels of markers of systemicinformation. It should be understood that the anti-inflammatory agentsof the invention are used to prevent cardiovascular disorders, that is,they are used prophylactically in subjects at risk of developing acardiovascular disorder. Thus, an effective amount is that amount whichcan lower the risk of, slow or perhaps prevent altogether thedevelopment of a cardiovascular disorder. When the agent is one thatbinds to cellular adhesion molecules and inhibits the ability of whiteblood cells to attach to such molecules, then the agent may be usedprophylactically or may be used in acute circumstances, for example,post-myocardial infarction or post-angioplasty. It will be recognizedwhen the agent is used in acute circumstances, it is used to prevent oneor more medically undesirable results that typically flow from suchadverse events. In the case of myocardial infarction, the agent can beused to limit injury to the cardiovascular tissue which develops as aresult of the myocardial infarction and in the case of restenosis theagent can be used in amounts effective to inhibit, prevent or slow thereoccurrence of blockage. In either case, it is an amount sufficient toinhibit the infiltration of white blood cells and transmigration ofwhite blood cells into the damaged tissue, which white blood cells canresult in further damage and/or complications relating to the injury.

Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50-500mg/kg will be suitable, preferably orally and in one or severaladministrations per day. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The anti-inflammatory agents may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the anti-inflammatory agent,which is preferably isotonic with the blood of the recipient. Thisaqueous preparation may be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. Oral administration will be preferred for prophylactictreatment because of the convenience to the patient as well as thedosing schedule.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing theanti-inflammatory agent into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing theanti-inflammatory agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the anti-inflammatory agent. Other compositionsinclude suspensions in aqueous liquids or non-aqueous liquids such as asyrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono-di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe anti-inflammatory agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) difusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

EXAMPLE

Study Organization

The Physicians' Health Study is a randomized, double-blind, placebocontrolled, 2×2 factorial trial of aspirin and beta-carotene in theprimary prevention of cardiovascular disease and cancer.

Subject Recruitment

A total of 22,071 US male physicians aged 40 to 84 years in 1982 with nohistory of myocardial infarction, stroke, transient ischemic attack, orcancer were assigned to one of four treatment groups: 325 mg aspirin onalternate days (Bufferin, provided by Bristol-Myers), 50 mg ofbeta-carotene on alternate days (Lurotin, provided by BASF Corporation),both, or neither. The aspirin component of the PHS was terminated earlyon Jan. 25, 1988 primarily due to a statistically extreme 44 percentreduction in risk of first infarction in the aspirin group.¹ Thebeta-carotene component continued to scheduled termination on Dec. 31,1995.²

Before randomization, between August 1982 and December 1984, potentiallyeligible participants were asked to provide baseline blood samplesduring a 16 week run-in period with all subjects given active aspirin.Blood collection kits including EDTA vacutainer tubes were sent toparticipants with instructions for taking blood. Participants were askedto have their blood drawn into the EDTA tubes, centrifuge the tubes, andreturn the plasma (accompanied by a provided cold pack) by overnightcourier. Upon return, specimens were alliquotted and stored at −80° C.Of 22,071 participants in the PHS, 14,916 (68 percent) provided baselineplasma samples. Over the 14 year period of the trial, no specimen hasinadvertently thawed during storage.

Endpoint Confirmation and Selection of Controls

Hospital records (and for fatal events, death certificates and necropsyreports) were requested for all reported cases of myocardial infarction,stroke, and venous thrombosis. Records were reviewed by a committee ofphysicians using standardized criteria to confirm or refute reportedevents. Endpoints reviewers were blinded to treatment assignment.

Reported myocardial infarction was confirmed if the event met WorldHealth Organization criteria of symptoms plus either elevated enzymes orcharacteristic electrocardiographic changes. Silent myocardialinfarctions were not included since they could not be dated accurately.Deaths due to coronary disease were confirmed based on autopsy reports,symptoms, circumstances of death, and prior history of coronary disease.Reported stroke was confirmed based on medical records showingneurological deficit of sudden or rapid onset persisting for more than24 hours or until death. Strokes were classified as ischemic orhemorrhagic. Computed tomography was available for more than 95 percentof confirmed strokes. Reported deep venous thrombosis was confirmed bydocumentation of a positive venography study or a positive ultrasoundstudy; deep venous thrombosis documented only by impedanceplethysmography or Doppler examination without ultrasound were notconfirmed. Reported pulmonary embolism was confirmed by positiveangiogram or completed ventilation-perfusion scan demonstrating at leasttwo segmental perfusion defects with normal ventilation.

Each participant who provided an adequate baseline plasma sample and hada confirmed myocardial infarction, stroke, or venous thrombosis afterrandomization was matched to one control. Controls were participatingphysicians who provided baseline plasma samples and reported nocardiovascular disease at the time the case reported his event. Controlswere randomly selected from study participants who met the matchingcriteria of age (+/− one year), smoking habit (current, past, or never),and time since randomization (six month intervals). Using these methods,we evaluated 543 cases and 543 controls in this prospective nestedcase-control design.

Collection of Plasma Samples and Laboratory Analysis

For each case and control, plasma collected and stored at baseline wasthawed and assayed for C-reactive protein employing enzyme linkedimmunoabsorbant assays (ELISA) based upon purified protein andpolyclonal anti-protein antibodies (Calbiochem).³ In brief, antibodiesare used to coat microtiter plate wells, and biotinylated C-reactiveprotein plus patient plasma is diluted 1:700 in assay buffer(phosphate-buffered saline with 0.1 percent Tween-20, and 1 percentbovine serum albumin). After competition, excess is washed off and theamount of biotinylated protein estimated by the addition ofavidin-peroxidase (Vectastain, Vector Laboratories, Burlingame, Calif.).Purified proteins are then used as standards, with the proteinconcentrations as determined by the manufacturer. The C-reactive proteinassay was standardized using the 1st International Reference Standard ofthe World Health Organization and has sensitivity to 0.08 ug/microliterwith standard reference range between 0.5 and 2.5 mg/liter. Methods usedto measure total and HDL cholesterol, triglyceride, lipoprotein(a),total plasma homocysteine, fibrinogen, D-dimer, and endogenoustissue-type plasminogen activator (tPA) antigen have been describedelsewhere.⁴⁻⁸

Blood specimens were analyzed in blinded pairs with the position of thecase varied at random within pairs to reduce the possibility ofsystematic bias and decrease interassay variability. The meancoefficient of variation for C-reactive protein across assay runs was4.2 percent.

Statistical Analysis

Means or proportions for baseline risk factors were calculated for casesand controls. The significance of any difference in means was testedusing the Student's t-test and the significance of any differences inproportions tested using the Chi square statistic. Because C-reactiveprotein levels are skewed, median levels were computed and thesignificance of any differences in median values between cases andcontrols assessed using Wilcoxon's Rank Sum Test. Geometric meanC-reactive protein levels were also computed after log transformationwhich resulted in near normal distribution. Tests for trends were usedto assess any relationship of increasing levels of C-reactive proteinwith risks of future vascular disease after dividing the sample intoquartiles defined by the distribution of the control values. Adjustedestimates were obtained using conditional logistic regression modelsaccounting for the matching variables and controlling for randomizedtreatment assignment, body mass index, diabetes, history ofhypertension, and a parental history of coronary artery disease. Similarmodels were employed to adjust for measured baseline levels of total andHDL cholesterol, triglyceride, lipoprotein(a), tPA antigen, fibrinogen,D-dimer, and homocysteine. To evaluate whether aspirin affected theserelationships, analyses were repeated for all myocardial infarctionevents occurring on or before Jan. 25, 1988, the date of termination ofrandomized aspirin assignment. All P values were two-tailed andconfidence intervals calculated at the 95 percent level.

Results

Table 1 shows baseline characteristics of study participants. Asexpected, those who subsequently developed myocardial infarction weremore likely than those who remained free of vascular disease to have ahistory of hypertension, hyperlipidemia, or a parental history ofcoronary artery disease. Similarly, those who subsequently developedstroke were more likely to be hypertensive. Due to the matching, age andsmoking were similar in cases and controls.

TABLE 1 Baseline Characteristics of Study Participants CardiovascularDisease During Follow-up None Any MI CVA DVT/PE (N = 543) (N = 543) (N =246) (N = 196) (N = 101) Age (yrs*) 59 +/− 9.1 59 +/− 9.2 58 +/− 8.6 62+/− 9.1 57 +/− 9.4 Smoking Status (%) Never 44 44 45 42 50 Past 41 41 4040 44 Current 15 15 15 18 6 Diabetes (%) 4 7 5 12 2 Body Mass Index(kg/m2*) 25 +/− 2.8 26 +/− 3.2 26 +/− 3.3 25 +/− 3.2 26 +/− 2.9 Historyof high cholesterol (%) 9 13 17 10 7 History of Hypertension (%) 16 2927 35 20 Parental history of 10 13 17 11 8 coronary artery disease (%)*values represent means +/− SD

Geometric mean and median levels of baseline C-reactive protein weresignificantly higher among those who subsequently developed any vascularevent compared to those who did not (P<0.001). The difference betweencases and controls was greatest for those who subsequently developedmyocardial infarction (1.51 mg/liter vs 1.13 mg/liter, P<0.001) althoughdifferences were also significant for stroke (P=0.03), particularlythose of ischemic etiology (P=0.02). In contrast, C-reactive proteinlevels were not significantly increased among those who subsequentlydeveloped venous thrombosis (P=0.34) (Table 2).

TABLE 2 Baseline levels of C-reactive protein among study participantswho remained free of vascular disease during follow-up (controls) andamong those who developed myocardial infarction, stroke, or venousthrombosis (cases) Baseline Level of C-Reactive Protein (mg/liter)Cardiovascular Disease Geometric During Follow-up Mean p Median p None(N = 543) 1.10 — 1.13 — Any Vascular Event (N = 246) 1.37 <0.001 1.40<0.001 Myocardial Infarction (N = 246) 1.48 <0.001 1.51 <0.001 AnyStroke (N = 196) 1.30 0.03 1.36 0.03 Ischemic Stroke (N = 154) 1.36 0.011.38 0.02 Venous Thrombosis (N = 101) 1.24 0.22 1.26 0.34

Relative risks of developing first myocardial infarction increasedsignificantly with each increasing quartile of baseline C-reactiveprotein (P for trend across quartiles<0.001) such that men in thehighest quartile had risks of future myocardial infarction almost 3times greater than those in the lowest (relative risk=2.9, 95 percentconfidence interval 1.8 to 4.6, P<0.001) (Table 3). Similarly, men withthe highest baseline C-reactive protein levels had twice the risk ofdeveloping future ischemic stroke (relative risk=1.9, 95 percentconfidence interval 1.1 to 3.3, P=0.02). No significant associationswere observed for venous thrombosis. Findings were similar in analyseslimited to non-fatal events.

TABLE 3 Relative risks of future myocardial infarction, stroke, andvenous thrombosis according to baseline levels of C-reactive protein.Quartile of C-Reactive Protein (range, mg/liter) 1 2 3 4 (≦0.55)(0.56–1.14) (1.15–2.10) (≧2.11) p-trend Myocardial Infarction (totalcohort) RR 1.0 1.7 2.6 2.9 <0.001 95% Cl — 1.1–2.9 1.6–4.3 1.8–4.6 p —0.03 <0.001 <0.001 Myocardial Infarction (non-smokers) RR 1.0 1.7 2.52.8 <0.001 95% Cl — 1.0–2.8 1.5–4.1 1.7–4.7 p — 0.06 <0.001 <0.001Ischemic Stroke RR 1.0 1.7 1.9 1.9 0.03 95% Cl — 0.9–2.9 1.1–3.2 1.1–3.3p — 0.07 0.02 0.02 Venous Thrombosis RR 1.0 1.1 1.2 1.3 0.38 95% Cl —0.6–2.0 0.7–2.3 0.7–2.4 p — 0.78 0.51 0.42 95% Cl = 95 percentconfidence interval

To evaluate whether increased baseline levels of C-reactive protein wereassociated with early rather than late thrombosis, we stratified theanalysis of myocardial infarction by years of follow-up. The relativerisk of future myocardial infarction associated with the highestquartile of C-reactive protein (as compared to the lowest quartile)ranged between 2.4 for events occurring in the first two years offollow-up to 3.2 for events occurring 6 or more years into studyfollow-up (Table 4). Similarly, the relative risk of future myocardialinfarction associated with a one quartile change in C-reactive proteinwas stable over long time periods (FIG. 4).

TABLE 4 Relative risks of first myocardial infarction associated withthe highest quartile of baseline C-reactive protein compared to thelowest quartile, according to year of study follow-up. Follow-Up Time(years) 0–2 2–4 4–6 6+ Total Cohort RR 2.4 2.9 2.8 3.2 95% Cl 0.9–6.81.1–7.6 1.1–6.9 1.2–8.5 p 0.09 0.03 0.03 0.02 Non-Smokers RR 2.8 2.9 2.72.9 95% Cl 0.9–8.7 1.0–8.3 1.0–7.0 1.1–8.2 p 0.07 0.05 0.05 0.04 95% CI= 95 percent confidence interval

Smokers had significantly higher median levels of C-reactive proteinthan non-smokers (2.20 mg/liter vs 1.19 mg/liter, P<0.001). Because ofthe match by smoking status, we minimized the potential for confoundingby smoking. However, to assess for effect modification, we repeatedanalyses limiting the cohort to non-smokers. As also shown in Table 3,the relative risks of future myocardial infarction among non-smokerssignificantly increased with each increasing quartile of C-reactiveprotein (P-trend<0.001). Similarly, the long term effects of C-reactiveprotein on risk of myocardial infarction were virtually identical amongnon-smokers (Table 4).

The relationship between C-reactive protein and myocardial infarctionwas not significantly altered in analyses which adjusted for body massindex, diabetes, hypertension, a family history of premature coronaryartery disease, total cholesterol, HDL cholesterol, triglycerides,lipoprotein(a), tPA antigen, D-dimer, fibrinogen, or homocysteine (Table5).

TABLE 5 Relative risks* of future myocardial infarction according tobaseline levels of C-reactive protein, adjusted for lipid and non-lipidvariables. Quartile of C-Reactive Protein (range, mg/liter) 1 2 3 4Variable(s) Adjusted for: (≦0.55) (0.56–1.14) (1.15–2.10) (≧2.11)p-trend Total and HDL Adjusted RR 1.0 1.8 2.2 2.3 0.002 Cholesterol 95%Cl — 1.0–3.1 1.3–3.7 1.4–3.9 p — 0.05 0.004 0.002 Triglyceride LevelAdjusted RR 1.0 1.8 2.1 2.8 <0.001 95% Cl — 1.0–3.2 1.2–3.7 1.6–4.9 p —0.06 0.008 <0.001 Lipoprotein(a) Adjusted RR 1.0 2.0 2.5 2.5 <0.001 95%Cl — 1.2–3.4 1.5–4.2 1.5–4.2 p — 0.01 <0.001 <0.001 tPA antigen levelAdjusted RR 1.0 1.7 1.9 2.9 0.002 95% Cl — 0.9–3.4 1.0–3.6 1.5–5.6 p —0.13 0.06 0.002 Total plasma Adjusted RR 1.0 1.8 2.9 3.6 <0.001homocysteine level 95% Cl — 1.1–3.1 1.7–4.8 2.1–5.9 p — 0.02 <0.001<0.001 D-dimer level Adjusted RR 1.0 2.2 2.4 2.7 0.001 95% Cl — 1.2–4.11.3–4.2 1.5–4.7 p — 0.007 0.003 <0.001 fibrinogen level Adjusted RR 1.02.2 2.2 2.9 0.01 95% Cl — 1.1–4.7 1.0–4.4 1.4–5.9 p — 0.04 0.04 0.005Body mass index Adjusted RR 1.0 1.5 2.4 2.6 <0.001 (kg/m²), diabetes,95% Cl — 0.9–2.5 1.5–4.0 1.6–4.4 history of hypertension, p — 0.14<0.001 <0.001 and family history of premature CAD *All models furtheradjusted for randomized aspirin and beta-carotene assignment. RR =relative risk, 95% CI = 95 percent confidence intervals

To assess whether the beneficial effect of aspirin on myocardialinfarction varied according to baseline C-reactive protein level, werepeated these analyses for events occurring prior to Jan. 25, 1988, thedate of termination of the randomized aspirin treatment.

Risks of developing future myocardial infarction increased with eachincreasing quartile of C-reactive protein for men randomly assigned toeither aspirin or placebo, and rates of myocardial infarction were lowerin the aspirin group for all quartiles of C-reactive protein (FIG. 5).However, the magnitude of the beneficial effect of aspirin on preventingmyocardial infarction was directly related to baseline C-reactiveprotein level. Specifically, randomized aspirin assignment wasassociated with a large and statistically significant reduction in riskof myocardial infarction among men with baseline C-reactive proteinlevels in the highest quartile (risk reduction=55.7 percent, P=0.02).However, among those with baseline C-reactive protein levels in thelowest quartile, the reduction in risk associated with aspirin was farsmaller and no longer statistically significant (risk reduction=13.9percent, P=0.77). These effects were linear across quartiles such thatthe apparent benefit of aspirin diminished in magnitude with eachdecreasing quartile of inflammatory risk (FIG. 5). This finding remainedessentially unchanged after further adjustment for other coronary riskfactors and the interaction between assignment to the aspirin group andbaseline C-reactive protein level (treated as a log transformedcontinuous variable) was statistically significant (P=0.048).

Data from the Physicians Health Study also indicate that measures ofinflammation such as C-RP predict the future risk of developingperipheral arterial disease, another clinical manifestation of systemicatherosclerosis. For example, those with baseline levels of C-RP inexcess of 2.0 mg/liter had twice the risk of developing futureperipheral arterial disease as did those with lower levels. Moreover, inthese data, the risks of developing peripheral arterial disease severeenough to require surgical intervention was increased fourfold for thosewith the highest baseline levels of C-RP.

To evaluate whether C-reactive protein might be a predictor of risk overand above that associated with cholesterol levels, a series ofstratified analyses were further performed. In this regard, C-reactiveprotein was found to predict risk of future myocardial infarction amongthose with low as well as high levels of total cholesterol, and amongthose with low as well as high total cholesterol to HDL cholesterolratios. Finally, to investigate whether the effect of C-reactive proteinare addictive to that of cholesterol, we performed further analyses inwhich study subjects were characterized by tertile (low, middle, orhigh) of cholesterol as well as C-reactive protein. Similar analyseswere performed in which study subject were characterized by tertile ofthe total cholesterol to HDL cholesterol ratio. As shown in FIGS. 2 and3, the risks of myocardial infarction associate with C-reactive proteinappear addictive to that of lipid parameters alone.

The actual C-reactive protein levels for the tested population are showngraphically in FIG. 6. The log normalized C-reactive protein levels areshown in FIG. 7, which demonstrates clearly the normal bell curvedistribution in our population. The mean C-reactive protein was 1.75 andthe standard deviation was 2.2. The mean of the log C-reactive proteinwas about 0.1 and the standard deviation was about 1. The relativeability to produce future cardiovascular disorder of another marker ofsystemic inflammation, soluble intracellular adhesion molecule(sICAM-1), also was evaluated. Table 6 shows the relative risks (RR) offuture myocardial infarction according to baseline levels of s-ICAM-1. Astatistically significant association was observed. The relationshipbetween s-ICAM and myocardial infarction also is not significantlyaltered in analysis which adjusted for body mass index, diabetes, afamily history of premature coronary artery disease, hyperlipidemia, anda history of hypertension.

TABLE 6 Relative risks (RR) of future myocardial infarction according tobaseline levels of soluble intercellular adhesion molecule (sICAM-1)Quartile of sICAM-1 (range, ng/liter) 1 2 3 4 p- (≦193) (193–224)(225–259) (>259) trend Crude RR 1.0 0.8 1.0 1.5 0.01 Lipid Adjusted* RR1.0 0.7 0.9 1.5 0.01 Fully Adjusted** RR 1.0 1.1 1.0 1.9 0.01 *Matchedfor smoking and age, controlled for total and HDL cholesterol **Matchedfor smoking and age, controlled for history of hypertension,hyperlipidemia, body mass index, diabetes, and a family history ofpremature CAD 95% Cl = 95 percent confidence intervalDiscussion

These prospective data indicate that baseline C-reactive protein levelamong apparently healthy men predicts risk of first myocardialinfarction and ischemic stroke. Further, the risks of arterialthrombosis associated with C-reactive protein were stable over longperiods of time and were not modified by other factors includingsmoking, body mass index, blood pressure, total and HDL cholesterol,triglyceride, lipoprotein(a), tPA antigen, D-dimer, fibrinogen, orhomocysteine. In these data, the risk of future myocardial infarctionassociated with C-reactive protein appears to be addictive to thatassociated with either total cholesterol or the total cholesterol to HDLcholesterol ratio. In contrast, the benefit of aspirin in reducing riskof first myocardial infarction diminished significantly with decreasingC-reactive protein level, an intriguing finding as this agent hasanti-inflammatory as well as anti-platelet properties. Finally, therewas no significant association for venous thromboembolism suggestingthat the relationship of inflammation with vascular risk may be limitedto the arterial circulation. We also observed a significant associationbetween risk of future myocardial infarction and a second measure ofsystemic inflammation, sICAM-1.

Since blood samples were collected at baseline, we can exclude thepossibility that acute ischemia affected C-reactive protein levels.Further, the statistically significant associations observed werepresent among non-smokers indicating that the effect of C-reactiveprotein on vascular risk is not simply the result of cigaretteconsumption.^(9,10) Thus, our prospective data relating baselineC-reactive protein level to future risks of myocardial infarction andstroke among apparently healthy men greatly extends prior observationsfrom studies of acutely ill patients¹², patients with symptomaticcoronary disease¹¹, or those at high risk due primarily to cigaretteconsumption.⁹ Moreover, in these data, the effects of C-reactive proteinwere independent of a large number of lipid and non-lipid risk factors.

The mechanisms by which C-reactive protein is related toatherothrombosis are uncertain. Prior infection with Chlamydiapnuemoniae, Heliobacter pylori, Herpes simplex virus, or cytomegalovirusmay be a source of the chronic inflammation detected by C-reactiveprotein.¹³⁻¹⁹ It is also possible that C-reactive protein is a surrogatefor interleukin-6²⁰, a cellular cytokine associated with macrophage andmonocyte recruitment into atherosclerotic plaque.²¹ In addition,C-reactive protein can induce monocytes to express tissue-factor, amembrane glycoprotein important in the initiation of coagulation.²²Finally, it had been hypothesized that bronchial inflammation secondaryto smoking is responsible for associations seen in prior studiesrelating C-reactive protein to vascular risk.⁹ In this regard, ourobservation that the effect of C-reactive protein is present amongnon-smokers makes bronchial inflammation a less likely mechanism.Further, the finding that the effects of C-reactive protein are stableover long time periods suggests that acute effects on clotting areunlikely.

Our data regarding the interrelation of C-reactive protein and aspirinmerit careful consideration. In the Physicians' Health Study, aspirinreduced risks of first myocardial infarction by 44 percent.¹ The presentfindings indicate that the effect of aspirin on first myocardialinfarction was greatest among those with highest baseline C-reactiveprotein levels and that the benefit diminished significantly inmagnitude with decreasing concentration of this inflammatory marker.

Some conclusions may be drawn. First, among apparently healthy men,baseline level of inflammation as assessed by C-reactive proteinpredicts risk of first myocardial infarction and ischemic stroke,independent of other risk factors. Second, baseline C-reactive proteinlevel is not associated with venous thrombosis, a vascular eventgenerally not associated with atherosclerosis. Third, C-reactive proteinis not simply a short term marker as previously demonstrated forpatients with unstable angina¹², but also a long term marker of risk,even for events occurring after 6 or more years. This observationsuggests that the effects of inflammation are likely mediated through achronic process, and excludes the possibility that undetected acuteillness at baseline is responsible for observed effects. Fourth, thesedata suggest that assessment of C-reactive protein can add to ourability to predict atherosclerotic risk, over and above that defined bylevels of total cholesterol and the total cholesterol to HDL cholesterolratio. Finally, the benefits of aspirin appear to be modified byunderlying inflammation.

REFERENCES

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1. A method for treating a nonhypercholesterolemic human in need thereofto reduce the risk of a cardiovascular disorder associated withatherosclerotic disease, comprising first selecting the human on thebasis that the human is known to have an above-normal level ofC-reactive protein and on the basis that the human is known to benonhypercholesterolemic, and then administering to the human because thehuman has an above-normal level of C-reactive protein a statin lipidreducing agent in an amount effective to lower the risk of the humandeveloping a future cardiovascular disorder associated withatherosclerotic disease.
 2. The method of claim 1, wherein the subjectis an apparently healthy non-smoker.
 3. The method of claim 1 whereinthe above-normal level of C-reactive protein is above 1.75 mg/l.
 4. Themethod of claim 1, wherein the above-normal level of C-reactive proteinis above 2.0 mg/l.
 5. The method of claim 2, wherein the above-normallevel of C-reactive protein is above 1.75 mg/l.
 6. The method of claim2, wherein the above-normal level of C-reactive protein is above 2.0mg/l.
 7. The method of claim 1, wherein the statin lipid reducing agentis lovastatin, fluvastatin, simvastatin, atorvastatin, pravastatin, orcerivastatin.
 8. The method of claim 2, wherein the statin lipidreducing agent is lovastatin, fluvastatin, simvastatin, atorvastatin,pravastatin, or cerivastatin.
 9. The method of claim 1, wherein thecardiovascular disorder associated with atherosclerotic disease ismyocardial infarction.
 10. The method of claim 1, wherein thecardiovascular disorder associated with atherosclerotic disease isstroke.
 11. The method of claim 2, wherein the cardiovascular disorderassociated with atherosclerotic disease is myocardial infarction. 12.The method of claim 2, wherein the cardiovascular disorder associatedwith atherosclerotic disease is stroke.